Cancer staging depends both on evaluation of both the primary tumor and metastatic disease. In the management of many cancers such as prostate or head and neck squamous cell carcinoma (HNSCC), the extent of lymph node metastasis can often only be fully evaluated after the patient has undergone surgical removal of all anatomically susceptible lymph nodes for pathological examination. Therefore, development of molecularly targeted imaging for more accurate detection of metastatic nodes during initial disease staging and surgery would be one of the most effective means to improve accuracy in staging and minimize morbidity associated with unnecessary lymph node dissections.
Matrix metalloproteinases (MMPs) play crucial roles in cancer invasion and metastasis (Bauvois, B., Biochim Biophys Acta 1825, 29-36 (2012)). While other MMPs are also of interest, MMP-2 and -9 are currently the species with the best-established associations with tumor grade/poor prognosis and with relatively specific substrate sequences. Although MMP-2,-9 are also increased in inflammation/wound healing, absolute levels of these gelatinases in the head and neck have been used to differentiate between benign papillomas versus carcinoma of the larynx (Uloza, V. et al., Eur Arch Otorhinolaryngol 268, 871-878 (2011)). Increased MMP-2,-9 expression has been shown to correlate with cancer grade (Wittekindt, C. et al., Acta Otolaryngol 131, 101-106 (2011), the content of which is hereby expressly incorporated by reference in its entirety for all purposes) and decreased survival (Liu, W. W. et al., Otolaryngol Head Neck Surg 132, 395-400 (2005); Mallis, A. et al., Eur Arch Otorhinolaryngol 269, 639-642 (2012), the contents of which are hereby expressly incorporated by reference in their entireties for all purposes). In carcinoma of the tongue, increased MMP-2,-9 expression has been shown to correlate with incidence of lymph node metastases (Zhou, C. X. et al., Aust Dent J55, 385-389 (2010), the content of which is hereby expressly incorporated by reference in its entirety for all purposes). We have previously described Activatable Cell Penetrating Peptides (ACPPs), which rely on tumor-associated MMP-2,-9 to unmask the adhesiveness of Cell Penetrating Peptides (CPPs) (Olson, E. S. et al., Integrative Biology 1, 382-393 (2009); Aguilera, T. A. et al., Integrative Biology 1, 371-381 (2009), the contents of which are hereby expressly incorporated by reference in their entireties for all purposes). Using fluorescently labeled ACPPs, improved surgical margin detection, decreased residual tumor burden, and improved survival in animal models of melanoma and breast cancer (Nguyen, Q. T. et al., Proc Natl Acad Sci USA 107, 4317-4322 (2010)) is shown.
Thrombin is a serine protease and a key regulator of blood coagulation. It is responsible for the proteolytic cleavage and activation of multiple coagulation factors including Factor V, VIII, XI as well as fibrinogen and protein C (E. W. Davie and J. D. Kulman, Seminars in Thrombosis and Hemostasis 2006, 32 Suppl 1, 3-15; J. A. Huntington, Journal of Thrombosis and Haemostasis 2005, 3, 1861-1872). Thrombin also cleaves and activates protease activated receptors (PARs) which are highly expressed on platelets, endothelial cells, myocytes and neurons (Vu T. K., et al., Cell 1991, 64, 1057-1068; Coughlin S. R., Nature 2000, 407, 258-264; Gallwitz M., et al., PloS One 2012, 7, e31756). Thrombin is a major therapeutic target for thrombosis and stroke intervention/prevention through indirect inhibitors such as heparin or warfarin, hirudin (divalent) and argatroban (monovalent) (Spyropoulos A. C., Thrombosis Research 2008, 123 Suppl 1, S29-35; Nutescu E. A., et al., Cardiology Clinics 2008, 26, 169-187, v-vi).
In addition to its role in thrombosis and stroke (Chen B, et al., Stroke; a journal of cerebral circulation 2010, 41, 2348-2352; Liu D. Z., et al., Annals of neurology 2010, 67, 526-533; Xue M. and Del Bigio M. R., Stroke; a journal of cerebral circulation 2001, 32, 2164-2169; Nishino A., et al., Journal of Neurotrauma 1993, 10, 167-179), thrombin is reported as a relevant player in cardiovascular disease (Leger A. J., et al., Circulation 2006, 114, 1070-1077; Aikawa E., et al., Circulation 2007, 116, 2841-2850), renal injury (Gupta A., et al., Current drug targets 2009, 10, 1212-1226), and cancer (Garcia-Lopez M. T., et al., Current medicinal chemistry 2010, 17, 109-128).
A number of fluorophore labeled peptide probes are known in the art. Chen et al.7 describe zipper molecular beacons (ZMB) comprising an asymmetrical polyarginine/polyglutamate electrostatic “zipper” hairpin-linked fluorophore-quencher pair. However, Chen et al tested their probes only in protein-free buffers or conditioned media, not in animals. They had difficulty in getting the cleaved probes to dissociate from each other, probably because of the greater hydrophobicity of their donor (pheophorbide) and quencher (BHQ3) compared to ours (Cy5 and Cy7 respectively). In the absence of bulk tissues or high protein concentrations, the hydrophobicity of their dyes probably kept the cleavage fragments glued together. Because their quencher (BHQ3) was nonfluorescent, and they showed no lifetime measurements, they missed the specific spectroscopic signatures of enzyme-mediated cleavage. Furthermore, BHQ-3 has been shown to be too unstable and easily metabolized for in vivo imagine.
Activatable cell penetrating peptides (ACPPs) target various cargoes including fluorescent imaging agents to sites of protease activity in vivo (Jiang T., et al., PNAS U.S.A. 2004, 101, 17867-17872; Olson E. S., et al., Integrative Biology: Quantitative Biosciences from Nano to Macro 2009, 1, 382-393; Olson E. S., et al., PNAS U.S.A. 2010, 107, 4311-4316; Aguilera T. A., et al., Integrative Biology: Quantitative Biosciences from Nano to Macro 2009, 1, 371-381). ACPPs consist of a polycationic cell penetrating peptide attached to a cargo and a polyanionic inhibitory domain with a protease cleavable linker. Probe activation and cargo uptake depends on localized proteolysis of the linker sequence that connects the polyanionic and polycationic domains, which converts the probe to an adherent form. This method provides detection of spatially localized enzymatic activity in living tissues via accumulation of cleaved probe.
ACPPs have been previously reported that target MMPs (Jiang T., et al., PNAS U.S.A. 2004, 101, 17867-17872; Olson E. S., et al., Integrative Biology: Quantitative Biosciences from Nano to Macro 2009, 1, 382-393, the contents of which are hereby expressly incorporated by reference in their entireties for all purposes) and elastases (Whitney M., et al, The Journal of Biological Chemistry 2010, 285, 22532-22541, the content of which is hereby expressly incorporated by reference in its entirety for all purposes) to cancer. A thrombin activated ACPP with cleavage sequence (SEQ ID NO:1) DPRSFL, from the PAR1 receptor was recently reported for monitoring thrombin activation in atherosclerotic plaques (Olson E. S., et al., Integrative Biology: Quantitative Biosciences from Nano to Macro 2012, 4, 595-605, the content of which is hereby expressly incorporated by reference in its entirety for all purposes). This ACPP is efficiently cleaved by thrombin and accumulates in atherosclerotic plaques with increasing signal depending on plaque load. An optimized and more selective thrombin cleavable ACPP with a substrate sequence of (SEQ ID NO:2) PPRSFL has also been used to measure thrombin activation after brain injury (Chen B., et al., The Journal of Neuroscience: the Official Journal of the Society for Neuroscience 2012, 32, 7622-7631, the content of which is hereby expressly incorporated by reference in its entirety for all purposes).
Each of these ACPPs include a single fluorophore (Cy5) and therefore quantitative measurement required time to allow uncleaved peptide to wash out of the target tissue before contrast developed. Probes based on fluorescence dequenching have previously been used to detect thrombin activity during clot formation, but many factors other than enzyme activity also affect fluorescence intensity, and diffusion of the agent after cleavage limits signal intensity at the site of protease activation (Jaffer F. A., et al., Arteriosclerosis, Thrombosis, and Vascular Biology 2002, 22, 1929-1935; Tung C. H., et al., Chembiochem: a European Journal of Chemical Biology 2002, 3, 207-211, the contents of which are hereby expressly incorporated by reference in their entireties for all purposes).
Much work has been done with simpler FRET substrates lacking the polycationic and polyanionic domains characteristic of ACPPs, thus with a fluorescent donor and quencher linked by an enzyme-cleavable sequence8. Almost all this work has been done for in vitro enzyme assays, because this simple design has no inherent provision for hindering diffusion and washout of the cleavage product containing the dequenched donor fluorophore. Therefore loss of spatial resolution after cleavage is a great concern for in vivo imaging.